title: Dissecting the salt dependence of the Tus-Ter protein-DNA complexes by high-throughput differential scanning fluorimetry of a GFP-tagged Tus.
abstract: The analysis of the salt dependence of protein-DNA complexes provides useful information about the non-specific electrostatic and sequence-specific parameters driving complex formation and stability. The differential scanning fluorimetry of GFP-tagged protein (DSF-GTP) assay has been geared with an automatic Tm peak recognition system and was applied for the high-throughput (HT) determination of salt-induced effects on the GFP-tagged DNA replication protein Tus in complex with various Ter and Ter-lock sequences. The system was designed to generate two-dimensional heat map profiles of Tus-GFP protein stability allowing for a comparative study of the effect of eight increasing salt concentrations on ten different Ter DNA species at once. The data obtained with the new HT DSF-GTP allowed precise dissection of the non-specific electrostatic and sequence-specific parameters driving Tus-Ter and Tus-Ter-lock complex formation and stability. The major factor increasing the thermal resistance of Tus-Ter-lock complexes in high-salt is the formation of the TT-lock, e.g. a 10-fold higher Kspe was obtained for Tus-GFP:Ter-lockB than for Tus-GFP:TerB. It is anticipated that the system can be easily adapted for the study of other protein-DNA complexes.
authors.affiliation: Centre for Biodiscovery and Molecular Development of Therapeutics, James Cook University, Douglas, QLD 4811, Australia.
authors.forename: Morgane J J
authors.initials: MJ
authors.lastname: Moreau
authors.forename: Patrick M
authors.initials: PM
authors.lastname: Schaeffer
mesh_terms: D001483:Base Sequence; D004261:DNA Replication; D004269:DNA, Bacterial / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism*; D005470:Fluorometry; D049452:Green Fluorescent Proteins / Q000378:metabolism; D057166:High-Throughput Screening Assays; D011485:Protein Binding; D012965:Sodium Chloride / Q000494:pharmacology*
chemical_list: D004269:DNA, Bacterial; D029968:Escherichia coli Proteins; C489236:tus protein, E coli; D049452:Green Fluorescent Proteins; D012965:Sodium Chloride